Recombinant DNA technology and genetic engineering have made it routinely possible to introduce desired DNA sequences into plant cells to allow for the expression of proteins of interest. For commercially viable transformation events, however, obtaining desired levels of stable and predictable expression in important crops remains challenging.
One method of expressing heterologous genes at desired levels in crops involves manipulation of the regulatory mechanisms governing expression in plants. The regulation may be transcriptional or post-transcriptional and can include, for example, mechanisms to enhance, limit, or prevent transcription of the DNA, as well as mechanisms that limit or increase the life span of an mRNA after it is produced. The DNA sequences involved in these regulatory processes can be located upstream, downstream or even internally to the structural DNA sequences encoding the protein product of a gene.
To regulate transcription in a transgenic plant, various types of promoters may be employed. Promoters can be used to control the expression of foreign genes in transgenic plants in a manner similar to the expression pattern of the gene from which the promoter was originally derived. In general, promoters are classified in two categories: “Constitutive” promoters express in most tissues most of the time, while “regulated” promoters are typically expressed in only certain tissue types (tissue specific promoters) or at certain times during development (temporal promoters). Expression from a constitutive promoter is typically more or less at a steady state level throughout development. Genes encoding proteins with house-keeping functions are often driven by constitutive promoters. Examples of constitutively expressed genes in maize include actin and ubiquitin (Ubi).
Further improvements in transcription can be obtained in transgenic plants by placing “enhancer” sequences upstream (5′) of the promoter. Enhancer elements are cis-acting and increase the level of transcription of an adjacent gene from its promoter in a fashion that is relatively independent of the upstream position and orientation of the enhancer. Such sequences have been isolated from a variety of sources, including viruses, bacteria and plant genes. One example of a well characterized enhancer sequence is the octopine synthase (ocs) enhancer from the Agrobacterium tumefaciens, as described in U.S. Pat. Nos. 5,837,849, 5,710,267 and 5,573,932. This short (40 bp) sequence has been shown to increase gene expression in both dicots and monocots, including maize, by significant levels. Tandem repeats of this enhancer have been shown to increase expression of the GUS gene eight-fold in maize. It remains unclear how these enhancer sequences function. Presumably enhancers bind activator proteins and thereby facilitate the binding of RNA polymerase II to the TATA box. WO95/14098 describes testing of various multiple combinations of the ocs enhancer and the mas (mannopine synthase) enhancer which resulted in several hundred fold increase in gene expression of the GUS gene in transgenic tobacco callus.
The use of a specific promoter, with or without one or more enhancers, however, does not necessarily guarantee desired levels of gene expression in plants. In addition to desired transcription levels, other factors such as improper splicing, polyadenylation and nuclear export can affect accumulation of both mRNA and the protein of interest. Therefore, methods of increasing RNA stability and translational efficiency through mechanisms of post-transcriptional regulation are needed in the art.
With regard to post-transcriptional regulation, it is has been demonstrated that certain 5′ and 3′ untranslated regions (UTRs) of eukaryotic mRNAs play a major role in translational efficiency and RNA stability, respectively. For example, the 5′ and 3′ UTRs of tobacco mosaic virus (TMV) and alfalfa mosiac virus (AMV) coat protein mRNAs are known to enhance gene expression in tobacco plants. The 5′ and 3′ UTRs of the maize alcohol dehydrogenase-1 (adh1) gene are known to be involved in efficient translation in hypoxic protoplasts.
Experiments with various 5′ UTR leader sequences demonstrate that various structural features of a 5′ UTR can be correlated with levels translational efficiency. Certain 5′ UTRs have been found to contain AUG codons which may interact with 40S ribosomal subunits when it scans for the AUG codon at the initiation site, thus decreasing the rate of translation. (Kozak, Mol. Cell. Biol. 7:3438 (1987); Kozak, J. Cell Biol. 108, 209 (1989)). Further, the 5′ UTR nucleotide sequences flanking the AUG initiation site on the mRNA may have an impact on translational efficiency. If the context of the flanking 5′ UTR is not favorable, part of the 40S ribosomal subunits might fail to recognize the translation start site such that the rate of polypeptide synthesis will be slowed. (Kozak, J. Biol. Chem. 266, 19867-19870 (1991); Pain, Eur. J. Biochem. 236, 747-771 (1996)). Secondary structures of 5′ UTRs (e.g., hairpin formation) may also hinder the movement of 40S ribosomal subunits during their scanning process and therefore negatively impact the efficiency of translation. (Sonenberg et al., Nature 334:320 (1988); Kozak, Cell 44:283-292, (1986)). The relative GC content of a 5′ UTR sequence has been shown to be an indicator of the stability of the potential secondary structure, with higher levels of GC indicating instability. (Kozak, J. Biol. Chem. 266, 19867-19870 (1991). Longer 5′ UTRs may exhibit higher numbers of inhibitory secondary structures. Thus, the translational efficiency of any given 5′ UTR is highly dependent upon its particular structure, and optimization of the leader sequence has been shown to increase gene expression as a direct result of improved translation initiation efficiency. Furthermore, significant increases in gene expression have been produced by addition of leader sequences from plant viruses or heat shock genes. (Raju et al., Plant Science 94: 139-149 (1993)).
In addition to 5′ UTR sequences, 3′ UTR (trailer) sequences of mRNAs are also involved in gene expression. 3′ UTRs (also known as polyadenylation elements or adenylation control elements) are known to control the nuclear export, polyadenylation status, subcellular targeting and rates of translation and degradation of mRNA from RNases. In particular, 3′ UTRs may contain one or more inverted repeats that can fold into stem-loop structures which act as a barrier to exoribonucleases, as well as interact with proteins known to promoter RNA stability (e.g., RNA binding proteins). (Barkan et al., A Look Beyond Transcription: Mechanisms Determining mRNA Stability and Translation in Plants, American Society of Plant Physiologists, Rockville, Md., pp. 162-213 (1998)). Certain elements found within 3′ UTRs may be RNA destabilizing, however. One such example occurring in plants is the DST element, which can be found in small auxin up RNAs (SAURs). (Gil et al., EMBO J. 15, 1678-1686 (1996)). A further destabilizing feature of some 3′ UTRs is the presence of AUUUA pentamers. (Ohme-Takagi et al., Pro. Nat. Acad. Sci. USA 90 11811-11815 (1993)).
3′ UTRs have been demonstrated to play a significant role in gene expression of several maize genes. Specifically, a 200 base pair 3′ sequence has been shown to be responsible for suppression of light induction of the maize small m3 subunit of the ribulose-1,5-biphosphate carboxylase gene (rbc/m3) in mesophyll cells. (Viret et al., Proc Natl Acad Sci USA. 91 (18):8577-81 (1994)). In plants, especially maize, this sequence is not very well conserved. One 3′ UTR frequently used in genetic engineering of plants is derived from a nopaline synthase gene (3′ nos) (Wyatt et al., Plant Mol Biol 22(5):731-49 (1993)).
In certain plant viruses, such as alfalfa mosaic virus (AMV) and tobacco mosaic virus (TMV), their highly structured 3′ UTRs are essential for replication and can be folded into either a linear array of stem-loop structures which contain several high-affinity coat protein binding sites, or a tRNA-like site recognized by RNA-dependent RNA polymerases. (Olsthoorn et al., EMBO J 1; 18(17):4856-64 (1999); Zeyenko et al., 1994)).
However, there remains a need to identify additional 5′ and 3′ UTRs for their use in regulating expression of recombinant nucleic acids in transgenic plants because there are no optimal UTR sequences available for every application.